Magnetic nanocompositions for highly sensitive molecular and cellular enrichment, purification and detection

ABSTRACT

The present invention relates to a nanocomposition capable of capturing or enriching an analyte at a sub-nanogram level and methods thereof. The nanocomposition can comprise a nanostructure operably linked to an analyte-capturing member.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 61/618,778 filed on Mar. 31, 2012, which is incorporatedherein by reference in its entirety.

BACKGROUND

Enrichment, purification and detection of analytes in biological samplesat a sub-nanogram level have always been a challenge in research anddevelopment. Therefore, there are needs in continuing to develop novelsystems and methods to achieve these purposes.

SUMMARY OF THE INVENTION

One aspect of the present disclosure relates to nanocompositions capableof capturing or enriching an analyte at a sub-nanogram level. In certainembodiments, the nanocomposition comprises a nanostructure operablylinked to an analyte-capturing member.

In certain embodiments, the sub-nanogram level of an analyte is no morethan 100 ng, 10 ng, 1 ng or 0.1 ng. For example, the sub-nanogramincludes 0.01 ng, 0.02 ng. 0.03 ng, 0.04 ng, 0.05 ng, 0.06 ng, 0.07 ng,0.08 ng, 0.09 ng, 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7ng, 0.8 ng, 0.9 ng, 1.0 ng, or any ranges between any of above mentionedlevel (e.g., between 0.01 ng and 100 ng, 0.01 ng and 10 ng, 0.01 ng and1 ng, 0.01 ng and 0.1 ng).

In certain embodiments, the sub-nanogram level of an analyte is no morethan 1000 pM, 100 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM, 0.001 pM (=1 fM) or0.0001 pM. For example, the sub-nanogram includes 0.001 pM (=1 fM),0.002 pM. 0.003 pM, 0.004 pM, 0.005 pM, 0.006 pM, 0.007 pM, 0.008 pM,0.009 pM, 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM,0.08 pM g, 0.09 pM, 0.1 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8pM, 9 pM, 10 pM or any ranges between any of above mentioned level(e.g., between 0.0001 pM and 1000 pM, 0.0001 pM and 100 pM, 0.0001 pMand 10 pM, 0.0001 pM and 1 pM, 0.0001 pM and 0.1 pM, 0.0001 pM and 0.01pM, 0.0001 pM and 0.001 pM).

In certain embodiments, the sub-nanogram level of an analyte, in thecase when the analyte is a biological sample, is a single sample (e.g.,a cell), a plurality of samples (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200 samples).

In certain embodiments, the analyte specifically binds to theanalyte-capturing member. In certain embodiments, the analyte-capturingmember can be a Protein A, a Protein G, or an antigen binding member. Incertain embodiments, the analyte can be an antibody that specificallybinds to Protein A or Protein G, or an antigen that specifically bindsto the antigen-binding member. In certain embodiments, thenanocomposition may further comprise an analyte specifically bound tothe analyte capturing member.

In certain embodiments, the analyte is a cell, part of a cell orsubcellular structure, a virus, an antibody, a protein/peptide, anucleic acid; carbohydrate, lipid, a polymer, or a small organicmolecule; a ligand, a receptor, a guest molecule, a host molecule, acirculating tumor cell, or a circulating DNA.

In certain embodiments, the nanostructure is a porous nanostructurewhich has been disclosed in U.S. Prov. Appl. 61/589,777 and U.S. patentapplication Ser. No. 12/460,009, both of which are incorporated hereinin their entirety). In certain embodiments, the nanostructure comprisesat least one core nanoparticle embedded in or coated with a low densityporous 3-D structure or coating, which is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure.

In certain embodiments, the core nanoparticle comprises a nanoparticleor a cluster of nanoparticles. A single core nanoparticle may comprise aplurality or a cluster of mini-nanoparticles. The nanoparticles in thecluster may be made by the same composition, or different compositions.

In certain embodiments, the core nanoparticle comprises a magneticmaterial. In certain embodiments, the magnetic material isferromagnetic, ferrimagnetic, paramagnetic materials, orsuperparamagnetic. In certain embodiments, the magnetic material issuperparamagnetic iron oxide (SPIO).

In certain embodiments, the core nanoparticle may further comprises anon-SPIO nanoparticle. The non-SPIO nanparticles include, for example,metallic nanoparticles (e.g., gold or silver nanoparticles), a metaloxide nanoparticle, semiconductor nanoparticle (e.g., quantum dots withindividual or multiple components such as CdSe/ZnS, doped heavy metalfree quantum dots or other semiconductor quantum dots); polymericnanoparticles (e.g., particles made of one or a combination of PLGA(poly(lactic-co-glycolic acid), PCL (polycaprolactone), PEG (polyethylene glycol) or other polymers); siliceous nanoparticles; andnon-SPIO magnetic nanoparticles (e.g., MnFe2O4, SAF, and other types ofmagnetic nanoparticles).

The core nanoparticle has a diameter ranging from about 1 nm to about900 nm (preferable 1-50 nm, 2-40 nm, 5-20 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16nm, 17 nm, 18 nm, 19 nm, 20 nm in size).

In certain embodiments, the core nanoparticle has a shape of sphere,rod, tetrapod, pyramidal, multi-armed, nanotube, nanowire, nanofiber, ornanoplate.

In certain embodiments, the low density, porous 3-D structure refers toa structure with density at least 10 s times (e.g. 10 s times, 20 stimes, 30 s times, 50 s times, 70 s times, 100 s times, 1000 s times,10,000 times) lower than existing mesoporous materials (e.g. mesoporousmaterials having a pore size ranging from 2 nm to 50 nm). In certainembodiments, the low density, porous 3-D structure has a density of <1.0g/cc (e.g. from 0.01 mg/cc to 1000 mg/cc). In certain embodiments, thedensity is determined using dry mass of the 3-D structure divided by thetotal volume of such 3-D structure in an aqueous solution.

In certain embodiments, the low density, porous 3-D structure is highlyporous. Such low density structure further refers to a structure havingat least 40% to at least 99.9% (preferably 50% to 99.9%) of empty spaceor porosity in the structure. In certain embodiments, at least 80% ofthe pores having size of 1 nm to 500 nm in pore radius.

In certain embodiments, the low density, porous 3-D structure is astructure that can not be obviously observed or substantially invisibleunder transmission electron microscope, for example, even when thefeature size of the low density structure is in the 10 s or 100 snanometer range.

In certain embodiments, the low density, porous 3-D structure is made ofsilicon-containing molecules (e.g., silanes, organosilanes,alkoxysilanes, silicates and derivatives thereof). For example, thesilicon-containing molecules can be amino-propyl-trimethoxysilane,mercapto-propyl-trimethoxysilane, carboxyl-propyl-trimethoxysilane,amino-propyl-triethoxysilane, mercapto-propyl-triethoxysilane,carboxyl-propyl-triethoxysilane, Bis[3-(triethoxysilyl)propyl]-tetrasulfide, Bis-[3-(triethoxysilyl) propyl]-disulfide,aminopropyltriethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, Vinyltrimethoxysilane,Vinyl-tris(2-methoxyethoxy) silane, 3-methacryloxypropyltrimethoxysilane, 2-(3,4-epoxycyclohexy)-ethyl trimethoxysilane,3-glycidoxy-propyltriethoxysilane, 3-isocyanatopropyltriethoxysilane,3-cyanatopropyltriethoxysilane, and sodium silicates.

In certain embodiments, the low density, porous 3-D structure isassociated with the core nanoparticle via intra-molecular interaction(e.g. covalent bonds, metallic bonds, and/or ionic bonding) orinter-molecular interaction (e.g. hydrogen bond, and/or non covalentbonds).

In certain embodiments, the low density, porous 3-D structure is astable crosslinked coating with thickness ranging from 1 nm to 1000 nm(e.g. from 1 nm to 500 nm). In certain embodiments, the thickness of thelow density, porous 3-D structure is controllable, so is the number ofpayloads that could be carried.

In certain embodiments, the nanostructure is capable of carrying orbeing associated with one or more payloads. In certain embodiments, thepayloads to be carried or associated with the nanostructure include, butare not limited to, a detectable agent (e.g. a fluorescent molecule, achemo-luminescent molecule, a bio-luminescent molecule, a radioisotope,a MRI contrast agent, a CT contrast agent, an enzyme-substrate label,and/or a coloring agent), a targeting moiety (e.g. an antibody, anantigen, a ligand, an aptamer, a peptide, a nucleic acid, apolynucleotide, a polysaccharide, sugar, fatty acid, steroids,pyrimidines, and/or a hapten), a binding partner (e.g. antigen,antibody, receptor, ligand, DNA, RNA, peptide, aptamer, biotin, avidin,streptavidin, lectin, carbohydrate, Protein A, antibody Fc,desthiobiotin, and/or iminobiotin), a biological active agent (e.g.therapeutic agents, proteins, antibodies, peptides, nucleic acids,enzymes, thermal-responsive molecules, optical-responsive molecules,electronic-responsive molecules, magnetic-responsive molecules,pH-responsive molecules, enzymatic responsive molecules and/or chemicalcompounds), a drug, a therapeutic agent, a radiological agent, achemological agent, a small molecule drug, a biological drug (e.g.,peptides, proteins, antibodies, antigens, nucleic acids, aptamers andthe like) and combinations thereof, which can be used to image, detect,study, monitor, evaluate, screen a disease, condition, and/or relatedbiological event. In certain embodiments, the nanostructure comprises afirst payload and a second payload. In certain embodiments, a payloadcan be an analyte-capturing member.

Another aspect of the present disclosure relates to methods ofcapturing, enriching, purifying, detecting or measuring an analyte in asample at a sub-nanogram level, comprising the steps of: a) contactingthe sample with a nanocomposition disclosed herein to form a mixturesolution and allowing the binding of the analyte with thenanocomposition, b) applying a magnetic field to the mixture, and c)evaluating the presence of or absence of an analyte.

In certain embodiments, the method further comprises a step of removingliquid from the mixture solution in the presence of the magnetic field.

In certain embodiments, the method further comprises a step of washingthe nanocomposition in the presence of magnetic field after removing theliquid and before eluting so as to remove un-bounded sample orun-bounded nanocompositions.

In certain embodiments, the method further comprises a step of elutingthe analyte from the nanocomposition, and collecting the analyte fromthe nanocomposition in the presence of a magnetic field, for example, byapplying an elution buffer.

In certain embodiments, the method further comprises a step of analyzingthe eluate by detecting the presence of the analyte or quantifying theamount of the analyte.

In certain embodiments, an increase in the ratio and/or an increase innanostructure facilitates the separation, purification or isolation.

Another aspect of the present disclosure relates to methods ofdetermining whether a test agent in a sample specifically binds to ananalyte and determine the nature of the agent, comprising the steps of:a) contacting the sample containing an analyte with a nanocomposition toform a mixture solution and allowing the binding of the agent with thenanocomposition, b) applying a magnetic field to the mixture, and c)evaluating whether there is a test agent binding to the analyte.

In certain embodiments, the method further comprises a step of removingliquid from the mixture solution in the presence of the magnetic field.

In certain embodiments, the method further comprises a step of washingthe nanocomposition in the presence of magnetic filed after removing theliquid and before eluting.

In certain embodiments, the method further comprises a step of elutingthe test agent from the nanocomposition and analyte, and analyzing thenature of the test agent, if the agent is present in the sample andbinds to the analyte.

Another aspect of the present disclosure relates to a method ofselecting or capturing an analyte (e.g., a cell) comprising the steps ofdetermining and applying a ratio of nanocompositions per said analyteand mixing the nanocompositions with said analyte based on the ratio. Incertain embodiments, the method increases the chance and/or amount ofanalytes captured.

Another aspect of present disclosure relates to a method of selecting orcapturing an analyte (e.g., a cell) with a desired feature comprisingthe steps of mixing a nanocompositions with a sample, evaluating thenumber of nanocompositions binding to one said analyte, and selectingthe analyte having the desired feature; wherein the desired featureincludes more than or less than a certain number of nanocompositionbinding to one analyte.

DESCRIPTION OF THE DRAWINGS

FIG. 1. An exemplary TEM image of silanized Au nanoparticles with coresize of Au at ˜20 nm and hydrodynamic size ˜60 nm. No siliceous coatingis visible from the TEM.

FIG. 2. An exemplary TEM image of silanized quantum dots withnanoparticle core size of ˜6 nm and hydrodynamic size ˜200 nm. Thesiliceous coating is not obviously visible from the TEM.

FIG. 3. Exemplary TEM images of coated nanoparticles known in the art,in which the coatings are obviously observable under TEM.

FIG. 4. A schematic comparison of porous nanostructure incorporating aplurality of payloads (dots) with dense nanostructures (black network,right).

FIG. 5. Illustration of the molecular enrichment and purificationprocess using magnetic nanocomposition disclosed herein.

FIG. 6. Illustration of the discovery of novel molecules that bind tothe analyte—which binds to nanocomposition disclosed herein.

FIG. 7. An exemplary TEM image of porous nanostructure, in which thediameters of the large core nanoparticles are shown.

FIG. 8. Magnetic nanostructure-Protein A conjugates show high bindingspecificity and sensitivity. 0.02 ng of antibodies binding to Protein Acan be detected.

FIG. 9. Magnetic nanostructure-Protein A conjugates show high recoveryrates for protein (e.g., antibodies) binding to Protein A.

FIG. 10. Both magnetic nanostructure-Protein G (PG) and magneticnanoparticle-protein A (PA) conjugates show high recovery rates forprotein (e.g., antibodies) binding to Protein A or G).

FIG. 11. Magnetic nanostructure-Protein A conjugates show high bindingspecificity and sensitivity to EP20 antibody. After EP 20 antibody iseluted from the binding of Protein A in an elution solution, thesolution is transferred to a vial. ELISA (enzyme linked immunosorbentassay) test is then used to measure the presence and amount of EP20antibodies. As shown in FIG. 11, 0.01 ng of EP20 antibody (in 50 ulsolution) can be detected.

FIG. 12. Magnetic nanostructure-Protein A conjugates show high recoveryrates for EP20 antibody that specifically binds to Protein A. Thehorseradish Peroxidase reaction time is 20 minutes. The recovery ratesfor EP20 antibodies are 72%, 59%, 69% and 48%, relative to 0.1 ng(sample 1#), 0.05 ng (sample 2#), 0.02 ng (sample 3#), and 0.01 ng(sample 4#) of EP20 antibodies respectively. The overall recovery rateis about or more than 50%, even when only 0.01 ng of EP20 antibodies arein solution.

FIG. 13. Magnetic nanostructure-Protein A conjugates show high recoveryrates for EP20 antibody that specifically binds to Protein A. Thehorseradish Peroxidase reaction time is 30 minutes. The recovery ratesfor EP20 antibodies are 70%, 73%, 66% and 49%, relative to 0.1 ng(sample 1#), 0.05 ng (sample 2#), 0.02 ng (sample 3#), and 0.01 ng(sample 4#) EP20 antibodies respectively. The overall recovery rate isabout or more than 50%, even when only 0.01 ng of EP20 antibodies are insolution.

FIG. 14. Magnetic nanostructure-Protein A conjugates show highsensitivity and recovery rates for rabbit antibody that specificallybinds to Protein A. 10 ul of Magnetic-Protein A Nanoparticles was mixedwith 0.1, 0.05, 0.02, or 0.01 ng antibody in 50 ul total volume. Theoverall recovery rate is higher than 50%, even when only 0.01 ng (1.25pM) of rabbit antibodies are in solution.

FIG. 15. Magnetic nanostructure-Protein A conjugates show highsensitivity for rabbit antibody that specifically binds to Protein A. 10ul of Magnetic-Protein A Nanoparticles was mixed with 0.01 ng antibodyin 1.5 ml or 10 ml volume (corresponding to 41.7 fM or 6.25 fM inconcentration).

FIG. 16. Capture of target cells is enhanced with an increased amount ofnanoparticles although the analyte binding member remains in constantamount. Different percentage of cells have been captured by fixing cellnumber at 100 k and antibody quantity at 250 ng. It indicates that thepercentage of bound cells increases with the number of magneticnanoparticles. In another words, the magnetic nanoparticles can be usedto select a desired population of cells by adjusting the ratio of thenumber of the magnetic beads to the cells. For example, this propertycould be used to select antibody high producer cells, or specific cellswith a low thresh-hold of number of markers on surface.

FIG. 17 shows bright field images of three samples with different numberof nanoparticles per cell. It indicates that the number of nanoparticlesper cell can be controlled by the starting ratio of the number ofnanoparticles to the number of cells before incubation. These figuresshow the cells tagged with different average number of beads. Thesecells are magnetically captured to the side of a vial using a magnet,then re-dispersed in cell medium and imaged under microscope.

FIG. 18 shows fluorescent magnetic nanoparticles for high capacity cellpurification and concurrent labeling. The nanoparticles are streptavidincoated, then conjugated with biotinlyated-EpCAM antibody. Cell linesused are human breast cancer cell MCF-7. Cells are tagged withnanoparticles, as cells divide, number of nanoparticle per celldecreases, after 5-7 days, majority of cells contain no nanoparticles.

FIG. 19 shows magnetic nanoparticles can be used to capture and identifycirculating tumor cells from whole blood samples. The recoveryefficiency is high even for low number of cancer cells spiked into wholeblood. The H1650 cells are used to evaluate the recovery efficiencyherein.

FIG. 20 shows purity of magnetically separated cancer cells from wholeblood samples is almost 100%. The left figure shows the bright fieldimage of magnetically separated 400 k H1650 cells from whole blood. Theright figure shows the corresponding fluorescent cell image bypre-labeling these cells with CFSE staining.

DETAILED DESCRIPTION OF THE INVENTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, solid state chemistry, inorganicchemistry, organic chemistry, physical chemistry, analytical chemistry,materials chemistry, biochemistry, biology, molecular biology,recombinant DNA techniques, pharmacology, imaging, and the like, whichare within the skill of the art. Such techniques are explained fully inthe literature.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

The following embodiments are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to perform the methods and use the probes disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a compound” includes a plurality of compounds. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Nanocomposition

One aspect of the present disclosure relates to a nanocompositioncapable of capturing or enriching an analyte at a sub-nanogram level.

The term “analyte” as used herein refers to a sample that are desired tobe captured, enriched, purified, detected or quantified. Examples ofanalyte include, without limitation, a molecule, a chemical, a compound,a biological sample, a cell, a virus, a bacteria, an antibody, aprotein/peptide, a nuclei acid (DNA or RNA, fragment thereof, anoligonucleotide, complimentary to the capturing member nuclei acid);carbohydrate, lipid, a polymer, or a small organic molecule such as adrug; a ligand that specifically binds to a receptor as ananalyte-capturing member, a receptor that specifically binds to a ligandas an analyte-capturing member, a guest molecule that specificallyinteracts with a host molecule or vice versa.

In certain embodiments, the analyte is part of a cell or subcellularstructure, such as membrane proteins or markers of cells, vesicles orexosomes. The nanocompositions can access and/or bind the analyte cells,vesicles, or exosomes that are otherwise not accessible to other typesof large magnetic beads.

In certain embodiments, the term “sub-nanogram level” refers to no morethan 100 ng, 10 ng, 1 ng or 0.1 ng of an analyte. For example, thesub-nanogram includes 0.01 ng, 0.02 ng. 0.03 ng, 0.04 ng, 0.05 ng, 0.06ng, 0.07 ng, 0.08 ng, 0.09 ng, 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng,0.6 ng, 0.7 ng, 0.8 ng, 0.9 ng, 1.0 ng, or any ranges between any ofabove mentioned level (e.g., between 0.01 ng and 100 ng, 0.01 ng and 10ng, 0.01 ng and 1 ng, 0.01 ng and 0.1 ng).

In certain embodiments, the sub-nanogram level means no more than 1000pM, 100 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM, 0.001 pM (=1 fM) or 0.0001 pMof an analyte. For example, the sub-nanogram includes 0.001 pM (=1 fM),0.002 pM. 0.003 pM, 0.004 pM, 0.005 pM, 0.006 pM, 0.007 pM, 0.008 pM,0.009 pM, 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM,0.08 pM, 0.09 pM, 0.1 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8pM, 9 pM, 10 pM or any ranges between any of above mentioned level(e.g., between 0.0001 pM and 1000 pM, 0.0001 pM and 100 pM, 0.0001 pMand 10 pM, 0.0001 pM and 1 pM, 0.0001 pM and 0.1 pM, 0.0001 pM and 0.01pM, 0.0001 pM and 0.001 pM).

In certain embodiments, the sub-nanogram level of an analyte, in thecase when the analyte is a biological sample, is a single sample (e.g.,a cell), a plurality of samples (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200 samples). For instance, the anlyateto be enriched or captured is a cell, and the sub-nanogram level is asingle cell or a plurality of cells.

The nanocomposition used herein comprises a nanostructure operablylinked to an analyte-capturing member.

The term “operably linked” as used herein, includes embedding,incorporating, integrating, binding, attaching combining, cross-linking,mixing, and/or coating the analyte-capturing member to that thenanostructure. The analyte-capturing member can be operably linked tothe nanostructure through non-covalent association (e.g. hydrogen bonds,ionic bonds, van der Waals forces, and hydrophobic interaction) orcovalent binding. For example, the analyte-capturing member mixed withand/or incorporated onto the surface of the nanostructure, or can alsobe loaded to the pores of the nanostructure.

Analyte-Capturing Member

In certain embodiments, the analyte-capturing members (oranalyte-binding members) are molecules capable of capturing orspecifically binding to an analyte. “Capturing”, “binding” or“specifically binding” as used herein, means that a non-random bindinginteraction between two molecules. The specific binding can becharacterized by binding affinity (Kd), which is calculated as the ratioof dissociation rate to association rate (koff/kon) when the bindingbetween the two molecules reaches equilibrium. The dissociation rate(koff) measured at the binding equilibrium may also be used whenmeasurement of kon is difficult to obtain, for example, due toaggregation of one molecule. The analyte-binding affinity (e.g., KD orkoff) can be appropriately determined using suitable methods known inthe art, including, for example, Biacore (see, for example, Murphy, M.et al, Current protocols in protein science, Chapter 19, unit 19.14,2006) and Kinexa techniques (see, for example, Darling, R. J., et al,Assay Drug Dev. Technol., 2(6): 647-657 (2004)).

Examples of analyte-capturing members include Protein A; Protein G;antigen-binding members (e.g., antibodies or fragments thereof); nucleicacid (or a fragment of nucleic acid, an oligo nucleotide); or aprotein/peptide binding specifically to a molecule such as anotherprotein/peptide, an antibody, a piece of nucleic acid (DNA or RNA),carbohydrate, lipid, a polymer, or a small organic molecule such as adrug; a ligand (e.g., a peptide, small molecule, hormone, a drug, toxin,neurotransmitter) that specifically binds to a receptor, or a receptorthat specifically binds to a ligand, a chemical in a supermolecularstructure (e.g., host-guest chemistry complex such as ap-xylylenediammonium bound within a cucurbituril) whereas the chemicalis a host molecule (e.g., cyclodextrins, calixarenes, cucurbiturils,porphyrins, metallacrowns, crown ethers, zeolites, cyclotriveratrylenes,cryptophanes and carcerands) or a guest molecule (e.g., prostaglandin,itraconazole).

Protein A is an affinity ligand for an antibody having an immunoglobulinFc domain, and can be useful in purification of antibodies that arebased on human .gamma.1, .gamma.2, or .gamma.4 heavy chains (Lindmark etal., J. Immunol. Meth. 62:1-13 (1983)). Similarly, protein G isrecommended for specific binding to antibodies of all mouse isotypes andfor antibodies based on human .gamma 3 heavy chains (Guss et al., EMBOJ. 5:1567 1575 (1986)). Avidin (or streptavdin) and biotin canspecifically bind to each other to form strong and specific non-covalentassociation. An antigen binding member can be an antibody, an antibodyfragment or an antibody memetics, such as, for example, scFV, Fab, Fab′,Fv, single domain antibody, diabody, nanobody, domain antibody, dsFv, orcanelized antibody. The antibodies or fragments can be polyclonal,monoclonal, of animal origin (e.g., murine, rabbit, camel), of humanorigin (e.g., fully human), chimeric, humanized, variable regions, CDRs,ScFv, bispecific, diabody, or other forms of antibodies withantigen-binding capabilities. In certain embodiments, the antibodies orantigen-binding fraction thereof specifically binds to a biomarker orbiomarkers such as a specific antigen of a cancer cell or a stem cell ora cell of interest.

In certain embodiments, the analyte-capturing members may be physicallyabsorbed into the nanostructure or covalently linked to thenanostructure through functional groups disclosed herein. In certainembodiments, the analyte-capturing members are operably linked to thenanostructure on its surface, so that the analyte capturing memberswould be accessible by the analyte.

In certain embodiments, the analyte is a substance that specificallyinteracts or binds to the analyte-capturing member. Example of analytesinclude a sample, a biological sample, a cell (e.g. an antibodyproducing hybridoma cell, a circulating tumor cell, a cell expressing adisease marker, etc.), a virus, an antibody, a protein/peptide, anucleic acid (DNA or RNA, fragment thereof, an oligonucleotide,complimentary to the capturing member nucleic acid); carbohydrate,lipid, a polymer, or a small organic molecule such as a drug; a ligandthat specifically binds to a receptor as an analyte-capturing member, areceptor that specifically binds to a ligand as an analyte-capturingmember, a guest molecule that specifically interacts with a hostmolecule or vice versa, antibodies (e.g. those specifically bind toProtein A or G), antigens (e.g. those that specifically bind toantigen-binding members), nucleic acid (e.g. those that specificallybind to antibodies like in Chromatin immunoprecipitation), proteins andprotein complexes (e.g. those that in immunoprecipitation orco-immunoprecipitations.).

A nanostructure can be operably linked to a suitable amount of theanalyte capturing member. The ratio of the nanostructure to the analytecapturing member can be appropriately adjusted by people skilled in theart according to the specific needs. For example, the ratio of thenanostructures to the analyte capturing member can be increased fordetection of a low amount of the analyte.

In certain embodiments, the nanocomposition may further comprise ananalyte specifically bound to the analyte capturing member. In certainembodiments, the analyte can be eluted from the nanocomposition.

Nanostructure

The term “nanostructure” as used herein, refers to a particle having adiameter ranging from about 1 nm to about 1500 nm (e.g. from 1 nm to1200 nm, from 1 nm to 1000 nm, from 1 nm to 800 nm, from 1 nm to 500 nm,from 1 nm to 400 nm, etc.). In certain embodiments, the nanostructurecomprises a single particle or a cluster of particles. In certainembodiments, the nanostructure comprises a core nanoparticle and acoating. The core nanoparticle can be a single or a cluster ofparticles. The coating can be any coating known in the art, for example,a polymer coating such as polyethylene glycol, silane, andpolysaachrides (e.g. dextran and its derivatives).

The nanostructures provided herein have a magnetic property or contain amagnetic material. Suitable magnetic materials include, for example,ferrimagnetic or ferromagnetic materials (e.g., iron, nickel, cobalt,some alloys of rare earth metals, and some naturally occurring mineralssuch as lodestone), paramagnetic materials (such as platinum, aluminum),and superparamagnetic materials (e.g., superparamagnetic iron oxide orSPIO).

The magnetic material has magnetic property which allows thenanostructure to be pulled or attracted to a magnet or in a magneticfield. Magnetic property can facilitate manipulation (e.g., separation,purification, or enrichment) of the nanostructures using magneticinteraction. The magnetic nanostructures can be attracted to ormagnetically guided to an intended site when subject to an appliedmagnetic field, for example a magnetic field from high-filed and/orhigh-gradient magnets. For example, a magnet or a magnetic grid can beplaced in the proximity of the nanostructures so as to attract themagnetic nanostructures. Magnetic field can also be formed throughelectromagensim (e.g., a solenoid carrying current).

In certain embodiments, the nanostructure provided herein comprises amagnetic nanoparticle which comprises a magnetic material. For example,the magnetic nanoparticle of the nanostructure is a superparamagneticiron oxide (SPIO) nanoparticle.

The SPIO nanoparticle is an iron oxide nanoparticle, either maghemite(γ-Fe₂O₃) or magnetite (Fe₃O₄), or nanoparticles composed of bothphases. The SPIO can be synthesized with a suitable method and dispersedas a colloidal solution in organic solvents or water. Methods tosynthesize the SPIO nanoparticles are known in the art (see, forexample, Morteza Mahmoudi et al, Superparamagnetic Iron OxideNanoparticles: Synthesis, Surface Engineering, Cytotoxicity andBiomedical Applications, published by Nova Science Pub Inc, 2011). Inone embodiment, the SPIO nanoparticles can be made through wet chemicalsynthesis methods which involve co-precipitation of Fe²⁺ and Fe³⁺ saltsin the presence of an alkaline medium. During the synthesis, nitrogenmay be introduced to control oxidation, surfactants and suitablepolymers may be added to inhibit agglomeration or control particle size,and/or emulsions (such as water-in-oil microemulsions) may be used tomodulate the physical properties of the SPIO nanoparticle (see, forexample, Jonathan W. Gunn, The preparation and characterization ofsuperparamagnetic nanoparticles for biomedical imaging and therapeuticapplication, published by ProQuest, 2008). In another embodiment, theSPIO nanoparticles can be generated by thermal decomposition of ironpentacarbonyl, alone or in combination with transition metal carbonyls,optionally in the presence of one or more surfactants (e.g., lauric acidand oleic acid) and/or oxidatants (e.g., trimethylamine-N-oxide), and ina suitable solvent (e.g., dioctyl ether or hexadecane) (see, forexample, US patent application 20060093555). In another embodiment, theSPIO nanoparticles can also be made through gas deposition methods,which involves laser vaporization of iron in a helium atmospherecontaining different concentrations of oxygen (see, Miller J. S. et al.,Magnetism: Nanosized magnetic materials, published by Wiley-VCH, 2002).In certain embodiments, the SPIO nanoparticles are those disclosed in USpatent application US20100008862.

In certain embodiments, the nanostructure herein can be a non-SPIOnanoparticle.

The non-SPIO nanoparticles can be prepared or synthesized using suitablemethods known in the art, such as for example, sol-gel synthesis method,water-in-oil micro-emulsion method, gas deposition method and so on. Forexample, gold nanoparticles can be made by reduction of chloroauratesolutions (e.g., HAuCl₄) by a reducing agent such as citrate, or acetonedicarboxulate. For another example, CdS semiconductor nanoparticle canbe prepared from Cd(ClO₄)₂ and Na₂S on the surface of silica particles.For another example, II-VI semiconductor nanoparticles can besynthesized based on pyrolysis of organometallic reagents such asdimethyl cadmium and trioctylphosphine selenide, after injection into ahot coordinating solvent (see, e.g., Günter Schmid, Nanoparticles: FromTheory to Application, published by John Wiley & Sons, 2011). Dopedheavy metal free quantum dots, for example Mn-doped ZnSe quantum dotscan be prepared using nucleation-doping strategy, in which small-sizedMnSe nanoclusters are formed as the core and ZnSe layers are overcoatedon the core under high temperatures. For another example, polymericnanoparticles can be prepared by emulsifying a polymer in a two-phasesolvent system, inducing nanosized polymer droplets by sonication orhomogenization, and evaporating the organic solvent to obtain thenanoparticles. For another example, siliceous nanoparticles can beprepared by sol-gel synthesis, in which silicon alkoxide precursors(e.g., TMOS or TEOS) are hydrolyzed in a mixture of water and ethanol inthe presence of an acid or a base catalyst, the hydrolyzed monomers arecondensed with vigorous stirring and the resulting silica nanoparticlescan be collected. For another example, SAFs, a non-SPIO magneticnanoparticle, can be prepared by depositing a ferromagenetic layer oneach of the two sides of a nonmagnetic space layer (e.g., rutheniummetal), along with a chemical etchable copper release layer andprotective tantalum surface layers, using ion-bean deposition in a highvacuum, and the SAF nanoparticle can be released after removing theprotective layer and selective etching of copper.

The size of the nanoparticles ranges from 1 nm to 100 nm in size(preferable 1-50 nm, 2-40 nm, 5-20 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm,17 nm, 18 nm, 19 nm, 20 nm in size). The size of nanoparticles can becontrolled by selecting appropriate synthesis methods and/or systems.For example, to control the size of nanoparticles, synthesis ofnanoparticles can be carried out in a polar solvent which provides ionicspecies that can adsorb on the surface of the nanoparticles, therebyproviding electrostatic effect and particle-particle repulsive force tohelp stabilize the nanoparticles and inhibit the growth of thenanoparticles. For another example, nanoparticles can be synthesized ina micro-heterogeneous system that allows compartmentalization ofnanoparticles in constrained cavities or domains. Such amicro-heterogeneous system may include, liquid crystals, mono andmultilayers, direct micelles, reversed micelles, microemulsions andvesicles. To obtain nanoparticles within a desired size range, thesynthesis conditions may be properly controlled or varied to providefor, e.g., a desired solution concentration or a desired cavity range (adetailed review can be found at, e.g., Vincenzo Liveri, Controlledsynthesis of nanoparticles in microheterogeneous systems, Published bySpringer, 2006).

The shape of the nanoparticles can be spherical, cubic, rod shaped (see,e.g., A. Fu et al., Nano Letters, 7, 179-182 (2007)), tetrapo-shaped(see, e.g., L. Manna et al., Nature Materials, 2, 382-385 (2003)),pyramidal, multi-armed, nanotube, nanowire, nanofiber, nanoplate, or anyother suitable shapes. Methods are known in the art to control the shapeof the nanoparticles during the preparation (see, e.g., Waseda Y. etal., Morphology control of materials and nanoparticles: advancedmaterials processing and characterization, published by Springer, 2004).For example, when the nanoparticles are prepared by the bottom-upprocess (i.e. from molecule to nanoparticle), a shape controller whichadsorbs strongly to a specific crystal plane may be added to control thegrowth rate of the particle.

A single nanostructure may comprise a single nanoparticle or a pluralityor a cluster of mini-nanoparticles (A. Fu et al., J. Am. chem. Soc. 126,10832-10833 (2004), J. Ge et al., Angew. Chem. Int. Ed. 46, 4342-4345(2007), Zhenda Lu et al., Nano Letters 11, 3404-3412 (2011).). Themini-nanoparticles can be homogeneous (e.g., made of the samecomposition/materials or having same size) or heterogeneous (e.g., madeof different compositions/materials or having different sizes). Acluster of homogeneous mini-nanoparticles refers to a pool of particleshaving substantially the same features or characteristics or consistingof substantially the same materials. A cluster of heterogeneousmini-nanoparticles refers to a pool of particles having differentfeatures or characteristics or consisting of substantially differentmaterials. For example, a heterogeneous mini-nanoparticle may comprise aquantum dot in the center and a discrete number of gold (Au)nanocrystals attached to the quantum dot. When the nanoparticles areassociated with a coating (as described below), different nanoparticlesin a heterogeneous nanoparticle pool do not need to associate with eachother at first, but rather, they could be individually and separatelyassociated with the coating.

In certain embodiments, a nanostructure disclosed comprises a pluralityof nanoparticles. For example, the nanostructure contains 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100, 100 s or 1000 s nanoparticles.

In certain embodiments, the nanostructure provided herein furthercomprises a coating. At least one core nanoparticle can be embedded inor coated with the coating. Any suitable coatings known in the art canbe used, for example, a polymer coating and a non-polymer coating.

The coating interacts with the core nanoparticles through 1)intra-molecular interaction such as covalent bonds (e.g., Sigma bond, Pibond, Delta bond, Double bond, Triple bond, Quadruple bond, Quintuplebond, Sextuple bond, 3c-2e, 3c-4e, 4c-2e, Agostic bond, Bent bond,Dipolar bond, Pi backbond, Conjugation, Hyperconjugation, Aromaticity,Hapticity, and Antibonding), metallic bonds (e.g., chelatinginteractions with the metal atom in the core nanoparticle), or ionicbonding (cation π-bond and salt bond), and 2) inter-molecularinteraction such as hydrogen bond (e.g., Dihydrogen bond, Dihydrogencomplex, Low-barrier hydrogen bond, Symmetric hydrogen bond) and noncovalent bonds (e.g., hydrophobic, hydrophilic, charge-charge, orπ-stacking interactions, van der Waals force, London dispersion force,Mechanical bond, Halogen bond, Aurophilicity, Intercalation, Stacking,Entropic force, and chemical polarity).

In certain embodiments, the coating comprises a low density, porous 3-Dstructure, as disclosed in U.S. Prov. Appl. 61/589,777 and U.S. patentapplication Ser. No. 12/460,007 (all references cited in the presentdisclosure are incorporated herein in their entirety).

The low density, porous 3-D structure refers to a structure with densitymuch lower (e.g., 10 s times, 20 s times, 30 s times, 50 s times, 70 stimes, 100 s times) than existing mesoporous nanoparticles (e.g.,mesoporous nanoparticles having a pore size ranging from 2 nm to 50 nm).(A. Vincent, et. al., J. Phys. Chem. C, 2007, 111, 8291-8298. J. E. Lee,et. al., J. Am. Chem. Soc., 2010, 132, 552-557. Y.-S. Lin, et. al., J.Am. Chem. Soc., 2011, 133, 20444-20457. Z. Lu, Angew. Chem. Int. Ed.,2010, 49, 1862-1866.)

In certain embodiments, the low density, porous 3-D structure refers toa structure having a density of <1.0 g/cc (e.g., <100 mg/cc, <10 mg/cc,<5 mg/cc, <1 mg/cc, <0.5 mg/cc, <0.4 mg/cc, <0.3 mg/cc, <0.2 mg/cc, or<0.1 mg/cc) (for example, from 0.01 mg/cc to 10 mg/cc, from 0.01 mg/ccto 8 mg/cc, from 0.01 mg/cc to 5 mg/cc, from 0.01 mg/cc to 3 mg/cc, from0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to 1 mg/cc, from 0.01 mg/cc to0.8 mg/cc, from 0.01 mg/cc to 0.5 mg/cc, from 0.01 mg/cc to 0.3 mg/cc,from 0.01 mg/cc to 1000 mg/cc, from 0.01 mg/cc to 915 mg/cc, from 0.01mg/cc to 900 mg/cc, from 0.01 mg/cc to 800 mg/cc, from 0.01 mg/cc to 700mg/cc, from 0.01 mg/cc to 600 mg/cc, from 0.01 mg/cc to 500 mg/cc, from0.1 mg/cc to 800 mg/cc, from 0.1 mg/cc to 700 mg/cc, from 0.1 mg/cc to1000 mg/cc, from 1 mg/cc to 1000 mg/cc, from 5 mg/cc to 1000 mg/cc, from10 mg/cc to 1000 mg/cc, from 20 mg/cc to 1000 mg/cc, from 30 mg/cc to1000 mg/cc, from 30 mg/cc to 1000 mg/cc, from 30 mg/cc to 900 mg/cc,from 30 mg/cc to 800 mg/cc, or from 30 mg/cc to 700 mg/cc).

The density of 3-D structure can be determined using various methodsknown in the art (see, e.g., Lowell, S. et al., Characterization ofporous solids and powders: surface area, pore size and density,published by Springer, 2004). Exemplary methods include, Brunauer EmmettTeller (BET) method and helium pycnometry (see, e.g., Varadan V. K. etal., Nanoscience and Nanotechnology in Engineering, published by WorldScientific, 2010). Briefly, in BET method, dry powders of the testing3-D structure is placed in a testing chamber to which helium andnitrogen gas are fed, and the change in temperature is recorded and theresults are analyzed and extrapolated to calculate the density of thetesting sample. In helium pycnometry method, dry powders of the testing3-D structure are filled with helium, and the helium pressure producedby a variation of volume is studied to provide for the density. Themeasured density based on the dry power samples does not reflect thereal density of the 3-D structure because of the ultralow density of the3-D structure, the framework easily collapses during the drying process,hence providing much smaller numbers in the porosity measurement thanwhen the 3-D structure is fully extended, for example, like when the 3-Dstructure is fully extended in a buffer solution.

In certain embodiments, the density of the 3-D structure can bedetermined using the dry mass of the 3-D structure divided by the totalvolume of such 3-D structure in an aqueous solution. For example, drymass of the core particles with and without the 3-D structure can bedetermined respectively, and the difference between the two would be thetotal mass of the 3-D structure. Similarly, the volume of a coreparticle with and without the 3-D structure in an aqueous solution canbe determined respectively, and the difference between the two would bethe volume of the 3-D structure on the core particle in an aqueoussolution.

In certain embodiments, the porous nanostructure can be dispersed asmultiple large nanoparticles coated with the 3-D structure in an aqueoussolution, in such case, the total volume of the 3-D structure can becalculated as the average volume of the 3-D structure for an individuallarge nanoparticle multiplied with the number of the largenanoparticles.

For each individual large nanoparticle, the size (e.g., radius) of theparticle with 3-D structure can be determined with Dynamic LightScattering (DLS) techniques, and the size (e.g., radius) of the particlecore without the 3-D structure can be determined under TransmissionElectron Microscope (TEM), as the 3-D structure is substantiallyinvisible under TEM. Accordingly, the volume of the 3-D structure on anindividual large nanoparticle can be obtained by subtracting the volumeof the particle without 3-D structure from the volume of the particlewith the 3-D structure.

The number of large nanoparticles for a given core mass can becalculated using any suitable methods. For example, an individual largenanoparticle may be composed of a plurality of small nanoparticles whichare visible under TEM. In such case, the average size and volume of asmall nanoparticle can be determined based on measurements under TEM,and the average mass of a small nanoparticle can be determined bymultiplying the known density of the core material with the volume ofthe small particle. By dividing the core mass with the average mass of asmall nanoparticle, the total number of small nanoparticles can beestimated. For an individual large nanoparticle, the average number ofsmall nanoparticles in it can be determined under TEM. Accordingly, thenumber of large nanoparticles for a given core mass can be estimated bydividing the total number of small nanoparticles with the average numberof small nanoparticels in an individual large nanoparticle.

Alternatively, the low density, porous 3-D structure refers to astructure having 40%-99.9% (preferably 50% to 99.9%) of empty space orpores in the structure, where 80% of the pores having size of 1 nm to500 nm in pore radius.

The porosity of the 3-D structure can be characterized by the Gas/Vaporadsorption method. In this technique, usually nitrogen, at its boilingpoint, is adsorbed on the solid sample. The amount of gas adsorbed at aparticular partial pressure could be used to calculate the specificsurface area of the material through the Brunauer, Emmit and Teller(BET) nitrogen adsorption/desorption equation. The pore sizes arecalculated by the Kelvin equation or the modified Kelvin equation, theBJH equation (see, e.g., D. Niu et al., J. Am. chem. Soc. 132,15144-15147 (2010)).

The porosity of the 3-D structure can also be characterized by mercuryporosimetry (see, e.g., Varadan V. K. et al., supra). Briefly, gas isevacuated from the 3-D structure, and then the structure is immersed inmercury. As mercury is non-wetting at room temperature, an externalpressure is applied to gradually force mercury into the sample. Bymonitoring the incremental volume of mercury intruded for each appliedpressure, the pore size can be calculated based on the Washburnequation.

Alternatively, the low density, porous 3-D structure refers to astructure that has a material property, that is, the porous structure(except to the core nanoparticle or core nanoparticles) could not beobviously observed or substantially transparent under transmissionelectron microscope, for example, even when the feature size of the 3-Dstructure is in the 10 s or 100 s nanometer range. The term “obviouslyobserved” or “substantially transparent” as used herein means that, thethickness of the 3-D structure can be readily estimated or determinedbased on the image of the 3-D structure under TEM. The nanostructure(e.g., nanoparticles coated with or embedded in/on a low density porous3-D structure) can be observed or measured by ways known in the art. Forexample, the size (e.g., radius) of the nanostructure with the 3-Dstructure can be measured using DLS methods, and the size (e.g., radius)of the core particle without the 3-D structure can be measured underTEM. In certain embodiments, the thickness of the 3-D structure ismeasured as 10 s, 100 s nanometer range by DLS, but cannot be readilydetermined under TEM. For example, when the nanostructures providedherein are observed under Transmission Electron Microscope (TEM), thenanoparticles can be identified, however, the low density porous 3-Dstructure can not be obviously observed, or is almost transparent (e.g.,see FIGS. 1 and 2). This distinguishes the nanostructures providedherein from those reported in the art (see, FIG. 3) that comprisenanoparticles coated with crosslinked and size tunable 3-D structure,including the mesoporous silica nanoparticles or coating (see, e.g., J.Kim, et. al., J. Am. Chem. Soc., 2006, 128, 688-689; J. Kim, et. al.,Angew. Chem. Int. Ed., 2008, 47, 8438-8441). This feature also indicatesthat the low density porous 3-D structure provided herein has a muchlower density and/or is highly porous in comparison to other coatednanoparticles known in the art.

The porosity of the 3-D structure can be further evaluated by thecapacity to load different molecules (see, e.g., Wang L. et al., NanoResearch 1, 99-115 (2008)). As the 3-D structure provided herein has alow density, it is envisaged that more payload can be associated withthe 3-D structure than with other coated nanoparticles (see, e.g., FIG.4). For example, when 3-D structure is loaded with organic fluorophoressuch as Rhodamin, over 10⁵ Rhodamin molecules can be loaded to 3-Dstructure of one nanoparticle.

In certain embodiments, the low density structure refers to a structurecapable of absorbing or carrying a fluorescent payload whosefluorescence intensity is at least 100 fold of that of the freefluorescent molecule (e.g., at least 150 fold, 200 fold, 250 fold, 300fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold or 600 fold). Thefluorescence intensity of a loaded nanoparticle can be quantified underthe same excitation and emission wave lengths as that of the fluorescentmolecules. The fluorescence intensity of the loaded low densitystructure indicates the payload of the fluorescent molecule, and alsoindirectly reflects the porosity of the low density structure.

In certain embodiments, the low density, porous 3-D structure is made ofsilane-containing or silane-like molecules (e.g., silanes,organosilanes, alkoxysilanes, silicates and derivatives thereof).

In certain embodiments, the silane-containing molecule comprises anorganosilane, which is also known as silane coupling agent. Organosilanehas a general formula of R_(x)SiY_((4-x)), wherein R group is an alkyl,aryl or organofunctional group. Y group is a methoxy, ethoxy or acetoxygroup. x is 1, 2 or 3. The R group could render a specific function suchas to associate the organosilane molecule with the surface of the corenanoparticle or other payloads through covalent or non-covalentinteractions. The Y group is hydrolysable and capable of forming asiloxane bond to crosslink with another organosilane molecule. ExemplaryR groups include, without limitation, disulphidealkyl, aminoalkyl,mercaptoalkyl, vinylalkyl, epoxyalkyl, and methacrylalkyl, carboxylalkylgroups. The alkyl group in an R group can be methylene, ethylene,propylene, and etc. Exemplary Y groups include, without limitation,alkoxyl such as OCH₃, OC₂H₅, and OC₂H₄OCH₃. For example, theorganosilane can be amino-propyl-trimethoxysilane,mercapto-propyl-trimethoxysilane, carboxyl-propyl-trimethoxysilane,amino-propyl-triethoxysilane, mercapto-propyl-triethoxysilane,carboxyl-propyl-triethoxysilane, Bis[3-(triethoxysilyl)propyl]-tetrasulfide, Bis-[3-(triethoxysilyl) propyl]-disulfide,aminopropyltriethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, Vinyltrimethoxysilane,Vinyl-tris(2-methoxyethoxy) silane, 3-methacryloxypropyltrimethoxysilane, 2-(3,4-epoxycyclohexy)-ethyl trimethoxysilane,3-glycidoxy-propyltriethoxysilane, 3-isocyanatopropyltriethoxysilane,and 3-cyanatopropyltriethoxysilane.

Payloads

In certain embodiments, the nanocomposition may further comprise apayload, which is carried or associated with the nanostructure. Thepayloads to be carried or associated with the nanostructure include, butare not limited to, a detectable agent, a biological active agent, adrug, a therapeutic agent, a radiological agent, a chemological agent, asmall molecule drug, a biological drug (e.g., peptides, proteins,antibodies, antigens, nucleic acids, aptamers, and the like) andcombinations thereof, which can be used to image, detect, study,monitor, evaluate, screen, the analyte of interest. Payloads may bephysically absorbed into the porous structure or linked to the porousstructure through functional groups disclosed herein. Payloads hereinalso include analyte-capturing members that bind specifically to theanalyte of interest.

A detectable agent can be a fluorescent molecule, a chemo-luminescentmolecule, a bio-luminescent molecule, a radioisotope, a MRI contrastagent, a CT contrast agent, an enzyme-substrate label, and/or a coloringagent etc. Examples of fluorescent molecules include, withoutlimitation, fluorescent compounds (fluorophores) which can include, butare not limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies); Alexa® Fluor 350; Alexa® Fluor 405; Alexa® Fluor 500;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC;APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ;Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550;Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl;Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; BodipyTMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-XSE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N;Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1Ca²⁺ Dye; Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18Ca²⁺; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX);Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA;Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; CoumarinPhalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™;Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; cyclic AMPFluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine;Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2;Dapoxyl 3′ DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR(Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA(4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH);DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydrorhodamine 123(DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS;DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide;Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF(Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4;Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin;Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin;Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, highcalcium; Indo-1, low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline;Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; OregonGreen; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; OregonGreen™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen);PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; PhloxinB (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA;Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE];PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3;PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI);PYMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2;Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; RhodamineBG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine;Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine;R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; SevronBrilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B;Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene;Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H;Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole orange(interchelating dyes), fluorescent semiconductor nanoparticles,lanthanides or combinations thereof.

Examples of radioisotopes include, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H,¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ¹⁸F, ²⁰¹Tl, ⁶⁷Ga, ¹³⁷Cs and otherradioisotopes.

Examples of enzyme-substrate labels include, luciferases (e.g., fireflyluciferase and bacterial luciferase), luciferin,2,3-dihydrophthalazinedionesm, alate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like.

In certain embodiment, at least one payload can be associated with thenanostructure. For example, only one group of homogenous payloads areassociated with the nanostructure. For another example, the firstpayload and the second payload are associated with the nanostructure andcan be different.

In certain embodiments, the first payload is associated with thenanostructure in one area of the structure and the second payload isassociated with the nanostructure in another area of the structure sothat the nanostructure can be directional or oriented with respect tothe distribution of payloads in the nanostructure. Such site selectivemodification could be achieved by depositing the nanostructure on asubstrate, then partially coating the substrate with a protectivepolymer layer, such as poly(3-hexyl-thiophene) (P3HT) and poly(methylmethacrylate) that could be dissolved using certain solvents such aschloroform or pyridine after the site selective modification isfinished. (Liu H., et. al., nano letters, 2004, vol 4., 2397-2401). Asecond modification could be achieved after the protective layer isremoved and the unmodified nanostructure is exposed and further modifiedwith different payloads. Further, the nanostructure could be depositedon the substrate with bonding through cleavable molecules, for example,photo cleavable molecules such as photocleavable biotin amine reactivelabeling reagents (www.ambergen.com), then the nanostructure could bereleased after site selective modifications. In addition, with cleavablelinker molecule bound to the substrate, the nanostructures could bereleased after the first site selective modification (SSM1). SSM1 couldinclude both functional groups for linking the SSM1 modified area to asubstrate in the next site selective modification step and payloads forsignal generating, drugs or other functional purposes. The procedurecould be repeated for SSM2. By controlling the protective polymer layerthickness, 3rd, 4th or more site selective modification steps could becarried out to render the nanostructures multiple regions of differentpayloads.

Methods of Use

Another aspect of the present disclosure relates to methods ofcapturing, enriching, purifying, detecting or measuring analytes at asub-nanogram level. The methods comprise steps of contacting ananalyte-containing sample with a nanocomposition disclosed herein toform a mixture (e.g., in a solution) and allow the binding of an analytewith the nanocomposition mixture (e.g., analyte-nanocompositionconjugate whereas the nanocomposition binds to the analyte via ananalyte-capturing member), applying a magnetic field to the mixture, andeluting the analytes from the nanocomposition. The method furthercomprises the step of washing the mixture prior to elution.

Referring to FIG. 5, a nanocomposition is obtained by operably linkingthe nanostructure disclosed herein to Protein A (an analyte-capturingmember) and the nanocomposition is incubated with a sample in a solutioncomprising an antibody for a period of time (e.g. 2 hrs) to allowProtein A to fully capture or bind to the antibody. Then theantibody-bounded nanocomposition is attracted and separated from otherelements in the sample by applying a magnetic field to the solution in acontainer. In the presence of a magnetic field, the sample-containingsolution is removed and the antibody-nanostructures are attracted to themagnetic field and left in the container. In a further step, theantibody is separated from Protein A or from the nanocomposition byapplying the elution buffer to the container wherein the elution bufferis sufficient to separate the binding of the antibody to Protein A. Themagnetic field is applied then to attract nanostructure while theseparated, un-bounded antibodies are in the eluate.

In certain embodiments, the method further comprises a step of washingthe nanocomposition in the presence of magnetic field after removing theliquid and before eluting. As shown in FIG. 5, for example, afterremoving the liquid from the mixture solution, the nanocomposition isredispersed in suitable washing liquid and then separated again byapplying a magnetic field. The washing step can be carried outrepeatedly.

In certain embodiments, the method further comprises a step of analyzingthe eluate by detecting the presence of the analyte or quantifying theamount of the analyte at a sub-nanogram level. As shown in FIG. 5, forexample, the antibody binding to Protein A is eluted from thenanocomposition with an elution buffer. The eluate is collected andanalyzed with methods known in the art.

Another aspect of the present disclosure relates to a method ofselecting or capturing an analyte (e.g., a cell) comprising the steps ofdetermining and applying a ratio of nanocompositions per said analyteand mixing the nanocompositions with said analyte based on the ratio. Incertain embodiments, an increase in the ratio of nanocomposition to theanalyte can enhance the chance and/or amount of the analyte capturedfrom the sample. Such methods can also be useful when a trace amount ofanalyte is to be separated or captured, for example, for identificationof circulating tumor cells, circulating DNAs in whole blood sample, orcertain marker cells expressing an interested biomarker (e.g. a diseasecell surface marker, and a stem cell marker, etc.). People in the artcan test the recovery rate of the analyte using different ratios of thenanocompositions to the analyte, while fixing the amount of the analyte.

Another aspect of the present disclosure relates to a method ofevaluating the bounded nanocompositions per analyte and selecting theanalyte with a certain properties that allow them to bind to a desirednumber of nanocompositions to carry enough magnetic moment to respond toa magnetic pull down. For magnetic nanoparticles that need a certainnumber of nanoparticles per cell, for example, 10 or 20 nanoparticlesper cell to allow the cells to be magnetically pulled down, the analytebounded with more magnetic nanocompositions more than the threshold ofthe number of nanoparticles per analyte will be pulled down andmagnetically captured when a magnetic field is applied, the analytebounded with less number of magnetic nanoparticles will not bemagnetically pulled down. Such methods are useful for selecting analytes(i.e. cells) having a desired number of markers, for example, selectinghybridoma cells producing a high level of antibodies, or selecting acell expressing a high level of certain markers. For example, a cellpopulation with different expression level of a certain marker iscaptured with the nanocomposition when the number of surface markerlarger than a certain number. The number of surface marker on the cellsurface that allow magnetic capture could be determined by a specificdisease indication, for example, cancer vs. normal cells, then the ratioof magnetic nanoparticles vs. the total cell number could be adjusted toallow that specific disease cell portion with a certain level of surfacemarker to be magnetically pulled down.

Another aspect of the present disclosure relates to methods ofdetermining whether a test agent in a sample specifically binds to ananalyte and/or determining the nature of the test agent, comprising thesteps of: a) contacting the sample containing an analyte and possiblythe agent to be determined with a nanocomposition in a mixture solutionand allowing the binding of the agent with the analyte which binds tothe nanocomposition, b) applying a magnetic field to the mixturesolution, and c) evaluating the presence, the nature, and/or the amountof the agent binding to the analyte.

In certain embodiments, the method further comprises a step of removingliquid from the mixture solution in the presence of the magnetic field.

In certain embodiments, the method further comprises a step of washingthe nanocomposition in the presence of magnetic filed after removing theliquid and before eluting.

In certain embodiments, the method further comprises a step of elutingthe agent and the analyte from the nanocomposition, and analyzing thepresence, nature and/or quantity of the agent. The analyte and the testagent can be eluted separately under different conditions. The methodmay further comprise recovering the test agent from the eluate.

In certain embodiments, the method is particularly useful for detectingan agent or another protein or biological analyte that binds to a knowanalyte, in particular, in protein-protein interaction. Other agentsthat bind to the known analyte can be detected using the method hereinand analyzed method well known in the art (e.g., mass spectrum, SDS gel,Western blot, ELISA, or antibodies-based methods) (see FIG. 6).

Methods for Preparing the Composition

Another aspect of the present disclosure relates to methods of forming ananocomposition comprising a nanostructure operably linked to ananalyte-capturing member.

In certain embodiments, the analyte-capturing members may be mixed witha readily formed nanostructure, e.g., in solution, dispersion,suspension, emulsion etc, to allow incorporation of theanalyte-capturing members to the porous compartment of thenanostructure, or to allow conjugation of the analyte-binding members tothe functional groups on the nanostructure.

In certain embodiments, the analyte-binding members may be introducedduring or after the formation of the nanostructures. For example, whenthe nanostructure is formed through silanization process, theanalyte-binding members can be introduced to the silanization system, soas to allow the incorporation of the analyte-binding member into thenanostructure during the silanization process. For another example, fora nanostructure having a surface reactive group (such as streptavidin),the analyte-binding member comprises a binding partner to the reactivegroup (such as biotin) can be mixed with the nanostructure underconditions which facilitate the binding.

Methods for Preparing the Nanostructure

Another aspect of the present disclosure relates to methods of forming ananostructure comprising at least one core nanoparticle with a coating.For example, the nanostructure is formed by coating or surrounding oneor more core nanoparticle with a coating material such that theparticle(s) is or are embedded in the coating material. For anotherexample, the coating material is formed by crosslinking a precursor inthe presence of a core nanoparticle, so that the nanoparticle isembedded in the crosslinked coating material.

In certain embodiments, the method further comprises introducing one ormore functional groups within or on the surface of the nanostructure.The functional groups may be introduced during the formation of thecoating material. For example, during the cross-linking process,precursors containing such functional groups can be added, inparticular, during the ending stage of the cross-linking process. Thefunctional groups may also be introduced after the formation of thenanostructure, for example, by introducing functional groups to thesurface of the nanostructure by chemical modification. In certainembodiments, the functional groups are inherent in the nanostructure orin the coating material.

The functional groups serve as linkage between the nanostructure and theanalyte capturing member. Examples of the functional groups include, butare not limited to amino, mercapto, carboxyl, phosphonate, biotin,streptavidin, avidin, hydroxyl, alkyl or other hydrophobic molecules,polyethylene glycol or other hydrophilic molecules, and photo cleavable,thermo cleavable or pH responsive linkers.

In certain embodiments, the method further comprises purifying theobtained nanostructure product. The purification may include use ofdialysis, tangential flow filtration, diafiltration, or combinationsthereof.

Methods for Preparing the Nanostructure Having a Low-Density Porous 3-DStructure

Another aspect of the present disclosure relates to methods of forming ananostructure comprising at least one core nanoparticle withlow-density, porous 3-D structure. For example, the nanostructure isformed by coating or surrounding one or more core nanoparticle with lowdensity, porous 3-D structure such that the particle(s) is or areembedded in the 3-D structure.

The low-density, porous 3-D structure is formed by the depositing, orcovering of the surface of the core nanoparticle through the assembly orcross-linking of silane-containing or silane-like molecules. The lowdensity porous 3-D structure can be prepared by a silanization processon the surface of the core nanoparticles. Silanization process includes,for example, the steps of crosslinking silicon-containing or silane-likemolecules (e.g., alkoxysilanes such as amino-propyl-trimethoxysilane,mercapto-propyl-trimethoxysilane, or sodium silicate) under acidic orbasic conditions.

In certain embodiments, an acidic or a basic catalyst is used in thecrosslinking. Exemplary acid catalyst include, without limitation, aprotonic acid catalyst (e.g., nitric acid, acetic acid and sulphonicacids) and Lewis acid catalyst (e.g., boron trifluoride, borontrifluoride monoethylamine complex, boron trifluoride methanol complex,FeCl₃, AlCl₃, ZnCl₂, and ZnBr₂). Exemplary basic catalysts include, anamine or a quaternary ammonium compound such as tetramethyl ammoniumhydroxide and ammonia hydroxide.

The silanization process may include one or more stages, for example, apriming stage in which the 3-D structure starts to form, a growth stagein which a layer of siliceous structure is readily formed on the corenanoparticle and more are to be formed, and/or an ending stage in whichthe 3-D structure is about to be completed (e.g., the outer surface ofthe 3-D structure is about to be formed). During the silanizationprocess, one or more silane-containing molecules can be added atdifferent stages of the process. For example, in the priming stage,organosilanes such as aminopropyl trimethoxyl silane or mercaptopropyltrimethoxyl silane can be added to initiate the silanization on the corenanoparticle surface. For another example, silane molecules having feweralkoxy groups (e.g., only 2 alkoxy groups) can be added to the reactionat the growth stage of silanization. For another example, at the endingstage of silanization, organo silane molecules with one or a variety ofdifferent functional groups may be added. These functional groups can beamino, carboxyl, mercapto, or phosphonate group, which can be furtherconjugated with other molecules, e.g., hydrophilic agent, a biologicallyactive agent, a detectable label, an optical responsive group,electronic responsive group, magnetic responsive group, enzymaticresponsive group or pH responsive group, or a binding partner, so as toallow further modification of the 3-D structure in terms of stability,solubility, biological compatibility, capability of being furtherconjugation or derivation, or affinity to payload. Alternatively, thefunctional groups can also be a group readily conjugated with othermolecules (e.g., a group conjugated with biologically active agent, athermal responsive molecule, an optical responsive molecule, anelectronic responsive molecule, a magnetic responsive molecule, a pHresponsive molecule, an enzymatic responsive molecule, a detectablelabel, or a binding partner such as biotin or avidin).

To control the formation of low density siliceous structure, thepreparation further includes density reducing procedures such asintroducing air bubbles in the reaction or formation, increasingreaction temperature, microwaving, sonicating, vertexing, labquakering,and/or adjusting the chemical composition of the reaction to adjust thedegree of the crosslinking of the silane molecules. Without being boundto theory, it is believed that these procedures can help make thereaction medium homogeneous, well dispersed and promote the formation oflow density porous 3-D structure with increased voids or porosity.

In certain embodiments, the density reducing procedure comprisessonicating the reaction or formation mixture. The conditions of thesonicating procedure (e.g., duration) in the silanization process can beproperly selected to produce a desired porosity in the resulting lowdensity porous 3-D structure. For example, the sonicating can be appliedthroughout a certain stage of the silanization process. The duration ofsonicating in a silanization stage may last for, e.g., at least 1 hour,1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours. In certainembodiments, sonicating is applied in each stage of the silanizationprocess.

In certain embodiments, the density reducing procedures compriseintroducing at least one alcohol to the reaction. In certainembodiments, the alcohol has at least 3 (e.g., at least 4, at least 5 orat least 6) carbon atoms. For example, the alcohol may have 3, 4, 5, 6,7, 8, 9, 10, 11, 12 or more carbon atoms. In certain embodiments, thealcohol can be monohydric alcohols, or polyhydric alcohols. Illustrativeexamples of monohydric alcohols include, propanol, butanol, pentanol,hexyl alcohol, etc. Illustrative examples of polyhydric alcoholsinclude, propylene glycol, glycerol, threitol, xylitol, etc. In certainembodiments, the alcohol can have a saturated carbon chain or anunsaturated carbon chain. An alcohol having a saturated carbon chain canbe represented as C_(n)H_((2n+2))O in chemical formula. In certainembodiments, n is no less than 3, or no less than 4, or no less than 5(e.g., n=3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more). Alcohol with anunsaturated carbon chain has a double or a triple bond between twocarbon atoms. In certain embodiments, the alcohol can be a cyclicalcohol, for example, cyclohexanol, inositol, or menthol.

In certain embodiments, the alcohol can have a straight carbon chain(e.g., n-propyl alcohol, n-butyl alcohol, n-pentyl alcohol, n-hexylalcohol, etc) or a branched carbon chain (e.g., isopropyl alcohol,isobutyl alcohol, tert-butyl alcohol, etc). In certain embodiments, thealcohol is present in a volume fraction of about 30% to about 70% (e.g.,about 30% to about 70%, about 30% to about 60%, about 30% to about 55%,about 40% to about 70%, about 45% to about 70%, about 40% to about 60%).In certain embodiments, the alcohol is present in volume fraction ofaround 50% (e.g., around 45%, around 46%, around 47%, around 48%, around49%, around 50%, around 51%, around 52%, around 53%, around 54%, around55%, around 56%, around 57%, around 58%, around 59%, or around 60%).

In certain embodiments, the density reducing procedure comprisesintroducing air bubbles to the reaction. In certain embodiments, the airbubbles can be in constant presence during the reaction process. The airbubbles can be introduced to the reaction through any suitable methods,for example, by blowing bubbles to the reaction, or by introducing agas-producing agent to the reaction mixture.

Other experimental conditions can also be optimized to provide forformation of a desired low density porous 3-D structure. Suchexperimental conditions include, for example, the concentration of thecore nanoparticles, the concentration of the catalyst, the ratio of theconcentration of the catalyst to the core nanoparticle, the temperatureat which the low density siliceous structure is formed, or the molecularstructure of the organosilanes.

The thickness of the low density porous 3-D structure, which directlycorrelates to the size of the nanostructure, could be controlled (e.g.,from 1 nm to 1000 nm) by, for example, modifying the quantity of thesilane-containing molecules (e.g., trialkoxysilane or sodium silicate),the reaction time, and time lapse between reaction steps and such kindof reaction parameters.

The thickness of the 3-D structure can be about 1 to 5 nm thick. Incertain embodiments, the thickness can be about 1 to 10 nm thick. Incertain embodiments, the thickness can be about 1 to 20 nm thick. Incertain embodiments, the thickness can be about 1 to 30 nm thick. Incertain embodiments, the thickness can be about 1 to 40 nm thick. Incertain embodiments, the thickness can be about 1 to 50 nm thick. Incertain embodiments, the thickness can be about 1 to 60 nm thick. Incertain embodiments, the thickness can be about 1 to 100 nm thick. Incertain embodiments, the thickness can be about 1 to 500 nm thick. Incertain embodiments, the thickness can be about 1 to 1000 nm thick.

After the low-density, porous 3-D structure is formed on the surface ofthe core nanoparticle, the core nanoparticle is embedded in the 3-Dstructure. The resulting nanostructure can have a thickness (e.g., thelongest dimension of the nanostructure or a diameter if the structure isa sphere) of about 1 to 1000 nm, 1 to 100 nm, or 1 to 10 nm. In anotherembodiment, the nanostructure can have a diameter of about 1 to 30 nm.In another embodiment, the nanostructure can have a diameter of about500 nm. In another embodiment, the nanostructure can have a diameter ofabout 100 nm. In another embodiment, the nanostructure can have adiameter of about 50 nm. In another embodiment, the nanostructure canhave a diameter of about 30 nm. In another embodiment, the nanostructurecan have a diameter of about 10 nm.

Products by Process

Another aspect of the present disclosure relates to nanocompositionprepared by any of the methods provided herein. The nanocompositionprepared herein may be operably linked with one or moreanalyte-capturing members, using methods described herein and/orconventional methods known in the art. The nanocomposition prepared inthe present disclosure can be further characterized for the 3-Dstructure, such as density, porosity, surface areas, thickness etc. ofthe 3-D structure. Optionally, the analyte-capturing members may becharacterized as well, such as the amount of the analyte-capturingmember or the detectable signal of the analyte-capturing member.

EXAMPLES Example 1 Preparation of Nanoparticles of Gold andSemiconductor Quantum Dots with the Low Density Siliceous Structure

The low density siliceous structure is a versatile and flexible platformfor making biocompatible nanoparticles. For example, to incorporate goldnanoparticles into the siliceous structure, Au nanoparticles synthesizedin either water solution or organic solutions could be utilized.Briefly, Au was precipitated out at the sample vial bottom aftercentrifuge at 13 k rpm for 15 min, then silane molecules such asaminopropyltrimethoxysilane and TMAOH was added. The reaction solventwas adjusted using a higher number alcohol, such as butanol or proponol.Then the sample was sonicated for a few hours with constant blowing ofair bubbles, afterwards, PEG-silane, mercaptopropyltrimethoxysilane andaminopropyltrimethoxysilane were added, the sample was sonicated foradditional 2-3 hours. Afterwards, mixture of chlorotrimethylsilane,methanol, and TMAOH or other silane molecules that only have one alkoxylgroup connecting with the silicon atom were added to react with surfacesiloxyl groups presented on the surface of the already grown siliceousstructure. After additional sonicating and aging, stable nanoparticleswith the highly porous siliceous structure were collected and storedwithin physiological buffer solutions through centrifugal filtering,centrifugation, dialysis or any other solution exchange methods. Theresulting Au nanostructure was observed under TEM, and an exemplary TEMimage was shown in FIG. 1. The nanoparticle core size was about 20 nmand hydrodynamic size was about 60 nm. The siliceous coating was notobvious from the TEM.

Example 2 Preparation of Nanoparticles of Semiconductor Quantum Dotswith the Low Density Siliceous Structure

As another example, semiconductor quantum dots in the form of individualnanocrystal or nanocrystal clusters could also be incorporated withinthe highly porous/low density siliceous structure. For example, CdSe/ZnSnanoparticles in organic solvents such as chloroform, Toluene, or Hexanecould be precipitated out by adding methanol and then throughcentrifugation. The nanocrystal pellet was then re-dispersed inaminopropyltrimethoxysilane or mercaptopropyltrimethoxysilane.Afterwards, tetramethyl ammonium hydroxide was added. Then the reactionsolvent was adjusted using a higher number alcohol, such as butanol orproponol. After sonicating the sample for 1-4 hours and blowing airbubbles, small amount of aminopropyltrimethoxysilane,mercaptopropyltrimethoxysilane, polyethyleneoxidesilane and water wassubsequently added, and the sample then underwent sonication for another1 to 4 hours. Then, mixture of chlorotrimethylsilane, methanol, andTMAOH or other silane molecules that only have one alkoxyl groupconnecting with the silicon atom were added. This sample was thensonicated for another 1-4 hours, followed by overnight aging under mildshaking or vibration. The resulting nanoparticles with lowdensity/highly porous siliceous structure were transferred intophysiological buffer solutions by centrifugal filtering, centrifugation,dialysis or any other solution exchange methods. The resulting CdSe/ZnSnanostructure was observed under TEM, and an exemplary TEM image wasshown in FIG. 2. The nanoparticle core size was about 10 nm andhydrodynamic size was about 200 nm. The siliceous coating was notobvious from the TEM.

Example 3 Preparation and Characterization of Low Density MagneticParticles

Preparation of the Magnetic Porous Nanostructure:

Magnetic particles formed by clustering multiple small particles andthen being coated were prepared. The clustering happened with theaddition of a worse solvent for generating dispersed nanoparticles, suchas butanol or isopropanol, followed by the addition of the silanizationreagents to form the porous nanostructure under constant blowing of airbubbles. The magnetic porous nanostructure as prepared was observedunder TEM (FIG. 7). As shown in FIG. 7, each large core nanoparticlecomprised a cluster of small nanoparticles, and the coating wassubstantially invisible under TEM.

Characterization of Density of the Coating:

To calculate the density of the coating, both the dry mass and thevolume of the coating were characterized.

Since the magnetic particles had high magnetic response that they couldbe directly captured using a magnet. This allowed generation of dryparticles to measure the mass of the material. The dry mass of particlesbefore and after coating was quantified as follows. 200 ul of the coatedparticle solution was pipetted out into a centrifugal vial whose masswas pre-measured. Coated magnetic nanoparticles were captured to theside of the vial wall, and the supernatant was removed. The capturedparticles were washed with water. At the end, the particles absorbed tothe side wall were left to dry in the open vial under a fume hood. Themass of the vial with the dry coated particles were measured. The drycoated particle mass was calculated by subtraction of the mass of thevial from the mass of the vial with the dry coated particles inside. Tomeasure the mass of the particles before coating, uncoated particlescorresponding to the same amount of the magnetic material as in thecoated nanoparticles, assuming an 80% coating processing yield, wascaptured to the side of the vial, and dried. The dry mass of theparticles before coating was measured by subtraction of the mass of thevial from the mass of the vial with the dry uncoated particles inside.The mass of the coating was equal to the mass of the dry coatedparticles minus the dry mass of particles before coating.

TABLE 1 Average Core mass (n = 3) 0.67 mg Average Coating mass (n = 3)0.06 mg

The total volume of the coating was calculated using the number of largeparticles in the above mass multiplied by the volume of the coating ofeach individual large nanoparticles. The particles were suspended in anaqueous solution, and the volume of the coating of each large particlewas calculated as 4/3×π(R³ _(with coating)−R³ _(core)), in which theR_(with coating) of an individual large nanoparticle was measured usingdynamic light scattering (DLS) technique, and the R_(core) of the largecore particle was directly imaged and measured using TEM (see FIG. 7).

TABLE 2 Average size of large core 210 nm nanoparticles under TEMAverage size of coated large 217-357 nm nanoparticles under DLS Averagecoating volume of an 4/3 × π (110³ − 105³) nm³ coated large nanoparticle

The number of large particles in the mass was calculated by dividing thetotal number of small nanoparticles by the number of small nanoparticlesin each large nanoparticle. The total number of small nanoparticles wasestimated by dividing the mass of total magnetic material by the mass ofan individual small nanoparticle (i.e. calculated using the size anddensity of the small nanoparticle). The number of small nanoparticles ineach individual large particle was counted from the TEM micrograph.Hence, the total volume of the coating can be calculated as the volumeof coating of a large nanoparticle multiplied by the total number of thelarge nanoparticles.

TABLE 3 Core mass 0.67 mg Density of core 5.2 kg/m³ Small nanoparticlesize 16 nm Small nanoparticle Volume 2.1 × 10⁻²⁴ m³ Mass of each smallnarnoparticle 1.1 × 10⁻¹⁷ mg Number of small particles in the core 6.1 ×10¹⁶ Average number of small particle per 236 large particle Number oflarge particles in the core 2.6 × 10¹⁴ Total volume of the coating 4/3 ×π (110³ − 105³) nm³ × 2.6 × 10¹⁴ = 0.1875 × 10⁻⁶ m³.

The density of the coating was calculated using the mass of the coatingdivided by the total volume of the coating, i.e., 0.06 mg/0.1875×10⁻⁶m³=0.32 mg/cm³.

The density of the low density siliceous structure prepared herein isonly 0.32 mg/cm³, which is significantly lower than the density of somereported silica coatings, for example, those reported in Vincent et al(Vincent, A. et al, J. Phys. Chem. C 2007, 111, 8291-8298), that have adensity of 1-2 g/cc and are 10⁴ denser than the siliceous structureprovided herein.

Characterization of Porosity Using BET Method:

Large magnetic nanoparticles after coating were captured to the side ofthe vial and dried. 2 samples of 65 mg (sample 1) and 45 mg (sample 2)dry mass were prepared for the BET measurement.

Surface pore sizes were measured using BET method for the dry mass ofthe coated nanoparticles. The results are shown in the below Tables.

TABLE 4 Characterization for Sample 1 Surface Area 14.166 m²/g Totalpore volume for pores 7.562 × 10⁻² cc/g smaller than 677.5 Å (Radius) atP/P₀ = 0.98562

TABLE 5 Characterization for Sample 2 Surface Area 6.380 m²/g Total porevolume for pores 7.099 × 10⁻² cc/g smaller than 683.3 Å (Radius) at P/Po= 0.98575

The surface area and the pore volume of the porous nanostructure weremeasured with dry mass of the porous nanostructure. If measured with aporous nanostructure sample suspended in an aqueous solution, the porevolume and the surface area are expected to be much higher than themeasurements with the dry mass, as the density of the coating has beenshown to be at least 10⁴ lower than those reported in the art.

The measured density based on the dry power samples does not reflect thereal density of the 3-D structure because of the ultralow density of the3-D structure, the framework easily collapses during the drying process,hence providing much smaller numbers in the porosity measurement thanwhen the 3-D structure is fully extended, for example, like when theporous nanostructure is fully extended in a buffer solution.

Example 4 Separation of an Antibody from a Sample

The magnetic porous nanostructures as prepared in Example 3 wereoperably linked to Protein A. Briefly, protein A was firstly activatedwith a crosslinker such as SMCC that could then be conjugated tonanoparticle surface. In general, the nanoparticle surfaces weresaturated with protein A molecules for maximal binding. The bindingcapacity of the magnetic nanoparticle-protein A conjugates could becharacterized. One method for such characterization involves mixingdifferent quantities of antibodies to the beads, followed by measuringthe amount of un-bound antibodies in the flow through with ELISA, thenthe quantity of bound antibody to beads could be calculated using thetotal quantity of antibody introduced minus the quantity of antibody inthe flow through. A saturation binding quantity of antibody could bederived from the plateau of the curve. The binding capacity ofantibodies of the magnetic nanoparticle-protein A samples range from 50ug/mg beads to 300 ug/mg beads based on the properties of antibody andthe measurement methods.

The protein A nanocomposition was washed twice in PBS-T by applying amagnetic field. The nanocomposition was collected and re-dispersed in 90μl of PBS-T, to which 10 μl of input antibody solution was further added(“Input”), containing 100 ng (Sample #2), 1000 ng (Sample #3) or 0.1 ng(Sample #4) antibody. A nanocomposition lacking protein A was used as acontrol (Sample #1), and was dispersed in 90 μl of PBS-T and was addedwith 10 μl of antibody solution containing 100 ng antibody. The mixtureof nanocomposition and the antibody was mixed well and incubated withrotating at room temperature for 2 hours. Afterwards, a magnet was usedto separate the magnetic nanocomposition from the solution, and theflowthrough (“FT”) was collected. Afterwards, the antibody captured bythe magnetic nanocomposition was eluted with 90 μl of IgG Elution Buffer(21009) for 1 min. 10 μl of 1M Tris (pH=8.0) was added in to neutralizethe solution and the final mixture was collected as elute (“Elute”).

Next, the quantities of antibodies in the input, flowthrough, and elutesolutions were quantified with ELISA. The ELISA plate was blocked with50 ul 1% BSA for 1 hour and washed with PBS-T three times. The samplescorresponding to Input, FT, and Elute were diluted 10-fold to 1 ng/μl.When the original quantity of antibody was less than 1 ng/μl, nodilution was performed. 5 μl of each sample and 45 μl of PBS were mixedand every mixture was incubated on an ELISA plate for 1 hour. Afterwashing with PBS-T for three times, the ELISA plate was incubated with50 μl of HRP (1:50 dilution) at room temperature for 1 hour. Then theELISA plate was washed with PBS-T for four times and 40 μl of TMBsubstrate was added. After 6 mins, the pictures of the ELISA plate weretaken (see FIG. 8, upper image) and the OD430 was recorded andnormalized to the input amount of the antibody. The test result wasshown in FIG. 8, lower image. Compared with the negative control (i.e.sample 1# with no Protein A), all the nanocompositions with protein Asuccessfully separated the antibody from the sample, even when theantibody was at a concentration of 0.1 ng.

In another experiment, the protein A nanocomposition was used toseparate an antibody. The nanocompositions were purified, re-dispersed,and incubated with the antibody, using the same procedure as describedabove. A magnet was used to separate the magnetic nanocomposition fromthe solution, and the flow through was collected. The nanocompositionwas washed with 100 μl of PBS-C once, and the wash was collected(“Wash”). The antibody captured by the nanocomposition was eluted withthe elution buffer, and the eluate was collected as described above. Asshown in FIG. 9, the antibody samples, although in differentconcentrations, were successfully separated with a recovery rate ofabove 90%. As compared with FIG. 8, the increase in the nanoparticleamount in this experiment resulted in significant increase in recoveryrate of the antibody. This indicated that, when separating an analyte inlow amount, increasing the amount of nanoparticles can increase therecovery rate.

In another experiment, both the protein A nanocomposition and theprotein G nanocomposition were used to separate an antibody. 10 ulnanocompositions were used to separate either 200 ng or 1000 ng antibodyin the sample. The separation was carried out in a similar procedure asdescribed above. As shown in FIG. 10, both protein A and protein Gmagnetic nanocompositions successfully separated the antibody from thesample, with a general recovery rate of above 80%.

Example 5 Purification of EP20 Antibody

The magnetic porous nanostructures as prepared in Example 3 wereoperably linked to Protein A, as described in Example 4.

The nanocompositions were purified, re-dispersed in 100 μl of PBS-T, andincubated with different amount of EP20 antibody (namely, 0.1 ng inSample 1, 0.05 ng in Sample 2, 0.02 ng in Sample 3, and 0.01 ng inSample 4), using the same procedure as described in Example 4. Theincubated nanocomposition was separated from the sample using a magnet,washed and then eluted. The flowthrough, the wash, and the elutesolutions were collected, and tested with ELISA as described above. Aphotograph of the ELISA plate was shown in FIG. 11. The resultsindicated that, the EP20 antibody, although at below an amount below 1ng, was successfully separated from the sample and eluted in the eluate.The presence of the antibody in the flowthrough or the wash was notobvious.

Example 6 Purification of Rabbit Antibody

The magnetic porous nanostructures as prepared in Example 3 wereoperably linked to Protein A, as described in Example 4.

The nanocompositions were purified, re-dispersed in 100 μl of PBS-T, andincubated with different amount of rabbit antibody (namely, 0.1 ng inSample 1, 0.05 ng in Sample 2, 0.02 ng in Sample 3, and 0.01 ng inSample 4), using the same procedure as described in Example 4. Theincubated nanocomposition was separated from the sample using a magnet,washed and then eluted. The flowthrough, the wash, and the elutesolutions were collected, and tested with ELISA as described above.These solutions were measured at 640 nm for absorbance, and the resultswere shown in FIGS. 12 and 13. The results indicated that, the rabbitantibody, although at an amount below 1 ng, was successfully separatedfrom the sample and eluted in the eluate. The recovery rate for theantibody at such a low amount was found to be at least 50%, and evenhigher.

Example 7 Purification of Rabbit Antibody in a Low Amount

The magnetic porous nanostructures as prepared in Example 3 wereoperably linked to Protein A, as described in Example 4.

The nanocomposition was were purified, re-dispersed in 10 μl of PBS-T,and incubated with different amount of rabbit antibody (namely, 0.1 ngin Sample 1, 0.05 ng in Sample 2, 0.02 ng in Sample 3, and 0.01 ng (1.25pM)) in Sample 4), in a total volume of 50 μl, using the same procedureas described in Example 4. The incubated nanocomposition was separatedfrom the sample using a magnet, washed and then eluted. The flowthrough,the wash, and the elute solutions were collected, and tested with ELISAas described above. These solutions were measured at 640 nm forabsorbance, and the results were shown in FIG. 14. The results indicatedthat, the rabbit antibody, although at below an amount below 1 ng and ina picomolar concentration, was successfully separated from the sampleand eluted in the eluate. The recovery rate for the antibody at such alow amount was found to be at least 50%, and even higher.

In another experiment, the nanocomposition was were purified,re-dispersed in 10 μl of PBS-T, and incubated with 0.01 ng of rabbitantibody in 1.5 ml (41.7 fM, Sample 1) or 10 ml volume (6.25 fM, Sample2), using the same procedure as described in Example 4. The incubatednanocomposition was separated from the sample using a magnet, washed andthen eluted. The flowthrough, the wash, and the elute solutions werecollected, and tested with ELISA as described above. These solutionswere measured at 640 nm for absorbance, and the results were shown inFIG. 15. The results indicated that, the rabbit antibody, although at anamount below 1 ng and in a femtomolar level, was successfully separatedfrom the sample and eluted in the elute. The recovery rate for theantibody at such a low amount demonstrated the high sensitivity of themethod.

Example 8 Selection of a Desired Population of Cells

Fluorescent magnetic nanoparticles were conjugated with streptavidin,and mixed with biotinylated antibody. A constant amount of 250 ngantibody was mixed with different amounts of the nanoparticles, i.e., at10 ul, 20 ul, 40 ul, and 80 ul, to prepare four different testcompositions. Then the four test compositions were added respectively tofour samples, each with a fixed cell number of 100 000 cells expressingthe antigen. The cells were incubated with the test compositions toallow capture of the cells by the antibody on the nanoparticles, andthen were separated by flowing through a magnetic grid. The magneticgrid was washed with PBS, and then eluted with an eluting buffer. Thesolution obtained after flowthrough (“FT”), washing (“Wash”) and eluting(“Bound”) were collected respectively, and detected for the amount ofcells (see FIG. 16). The percentage of captured cells increased with theincrease in the amount of the magnetic nanoparticles, although theamount of the antibody remained the same. The percentage of the cellswas up to 99.43% when 80 μl of magnetic nanoparticles was used, whereaswhen 10 μl of magnetic nanocomposition was added to the sample, thepercentage of captured cells was 32.69%.

The number of nanoparticles per cell could be controlled by the startingratio of the number of nanoparticles to cells before incubation. Cellswere mixed with magnetic nanoparticles at different ratios, namely, 7nanoparticles/cell, 25 nanoparticles/cell, and 89 nanoparticles/cell.These cells were magnetically captured to the side of a vial using amagnet, and then re-dispersed in cell medium and imaged undermicroscope. As shown in FIG. 17, by adjusting the starting ratio of thenumber of nanoparticles to cells, the bright field images showed thecells were tagged with different average number of beads. The cells ofdesired number of nanoparticle per cell are selected to represent a highpresence or expression of the analyte.

Example 9 Purification and Labelling of Cells

Fluorescent magnetic nanoparticles were coated with streptavidin andthen conjugated with biotinlyated-EpCAM antibody. Cell lines used herewere human breast cancer cell MCF-7. As shown in FIG. 18, the cells weretagged with nanoparticles, as cells divided, the number of nanoparticlesper cell decreased. After 5-7 days, majority of cells contained nonanoparticles.

Example 10 Recovery of Low Number of Cancer Cells Spiked

Fluorescent magnetic nanoparticles were coated with streptavidin andthen conjugated with biotinlyated-EpCAM antibody. 100, 20 or 4 H1650cells prestained with CFSE of green fluorescence were spiked into 1 mlwhole blood. The blood sample spiked with the H1650 cells were incubatedwith the magnetic nanoparticles, followed by magnetic pull-down with asmallmagnet. After removing the supernatant, the separated cells wereredispersed in medium and counted. As shown in FIG. 19, the recoveryefficiency was high even for low number of spiked H1650 cells.

In another experiment, 400,000 H1650 cells pre-labeled with CFSE ofgreen fluorescence were recovered from whole blood samples, usingmagnetic nanoparticles. As shown in FIG. 18, for each cell shown in thebright field image (left), there is a corresponding florescent cellimage (right), indicating that the cells recovered from the whole bloodare in high purity (almost 100%).

1. A nanocomposition comprising: a nanoparticle, and ananalyte-capturing member, wherein the nanoparticle is operably linked tothe analyte-capturing member, and the nanocomposition is capable ofcapturing an analyte at a sub-nanogram level.
 2. The nanocomposition ofclaim 1 wherein the sub-nanogram level of an analyte is no more than 100ng, 10 ng, 1 ng or 0.1 ng, or 0.01 ng.
 3. The nanocomposition of claim 1wherein the sub-nanogram level of an analyte is no more than 1000 pM,100 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM, 0.001 pM or 0.0001 pM.
 4. Thenanocomposition of claim 1 wherein the analyte specifically binds to theanalyte-capturing member.
 5. The nanocomposition of claim 1 wherein thenanoparticle contain a magnetic material.
 6. The nanocomposition ofclaim 5 wherein the magnetic material is ferromagnetic, ferrimagnetic,paramagnetic materials, or superparamagnetic.
 7. The nanocomposition ofclaim 6 wherein the magnetic materials is superparamagnetic iron oxide.8. The nanocomposition of claim 1 wherein the analyte-capturing memberis a Protein A, a Protein G, an antigen binding member, streptavidin,biotin or secondary antibody.
 9. The nanocomposition of claim 8 whereinthe analyte is an antibody that specifically binds to Protein A orProtein G, or an antigen that specifically binds to the antigen-bindingmember.
 10. The nanocomposition of claim 1 may further comprise ananalyte specifically bound to the analyte capturing member.
 11. Thenanocomposition of claim 1 wherein the analyte is a cell, part of a cellor subcellular structure, a virus, an antibody, a protein/peptide, anucleic acid; carbohydrate, lipid, a polymer, or a small organicmolecule; a ligand, a receptor, a guest molecule, a host molecule, acirculating tumor cell, or a circulating DNA.
 12. A method of capturing,enriching, purifying, detecting or measuring an analyte in a sample at asub-nanogram level, comprising the steps of a) contacting the samplewith a nanocomposition of claim 1 to form a mixture solution andallowing the binding of the analyte with the nanocomposition, b)applying a magnetic field to the mixture, and c) evaluating the presenceof or absence of the analyte.
 13. The method of claim 12 wherein thesub-nanogram level of an analyte is no more than 100 ng, 10 ng, 1 ng,0.1 ng or 0.01 ng.
 14. The method of claim 12 wherein the sub-nanogramlevel of an analyte is no more than 1000 pM, 100 pM, 10 pM, 1 pM, 0.1pM, 0.01 pM, 0.001 pM or 0.0001 pM.
 15. The method of claim 12 furthercomprising a step of removing liquid from the mixture solution in thepresence of the magnetic field.
 16. The method of claim 12 furthercomprising a step of washing the nanocomposition in the presence ofmagnetic field after removing the liquid and before eluting.
 17. Themethod of claim 12 further comprising a step of eluting the analyte fromthe nanocomposition, and collecting the analyte from the nanocompositionin the presence of a magnetic field.
 18. The method of claim 12 furthercomprising a step of analyzing the eluate by detecting the presence ofthe analyte or quantifying the amount of the analyte.
 19. The method ofclaim 12, further comprising determining and applying a ratio of thenanocomposition to the analyte and mixing the nanocomposition with thesample based on the ratio.
 20. The method of claim 12, furthercomprising evaluating the bounded nanocompositions per said analyte andselecting the analyte binding to a desired number of nanocompositions.21. A method of determining whether a test agent in a samplespecifically binds to an analyte, comprising the steps of a) contactinga sample containing an analyte with a nanocomposition of claim 1 to forma mixture solution and allowing the binding of the test agent with thenanocomposition, b) applying a magnetic field to the mixture, and c)evaluating whether there is a test agent binding to the analyte.
 22. Themethod of claim 21 wherein the test agent is at sub-nanogram level. 23.The method of claim 22 wherein the sub-nanogram level of the test agentis no more than 100 ng, 10 ng, Ing or 0.1 ng, or 0.01 ng.
 24. The methodof claim 22 wherein the sub-nanogram level of the test agent is no morethan 1000 pM, 100 pM, 10 pM, 1 pM, 0.1 pM, 0.01 pM, 0.001 pM (=1 fM) or0.0001 pM.
 25. The method of claim 21 further comprising a step ofremoving liquid from the mixture solution in the presence of themagnetic field.
 26. The method of claim 21 further comprising a step ofwashing the nanocomposition in the presence of magnetic field afterremoving the liquid and before eluting.
 27. The method of claim 21further comprising a step of eluting the test agent from thenanocomposition and the analyte, and collecting the test agent from thenanocomposition in the presence of a magnetic field.
 28. The method ofclaim 21 further comprising a step of analyzing the eluate by detectingthe presence of the test agent or quantifying the amount of the testagent.
 29. A method of determining the presence, nature, and/or quantityof a test agent in a sample that specifically binds to an analyte, or ispart of analyte (i.e., surface markers on cancer cell surfaces),comprising the steps of: a) contacting the sample containing an analytewith a nanocomposition of claim 1 in a mixture solution, b) allowing thebinding of the analyte to the nanocomposition, c) applying a magneticfield to the mixture solution, and d) evaluating the presence, thenature, and/or the amount of the agent binding to the analyte.